Int. J. Mol. Sci. 2015, 16, 256-283; doi:10.

3390/ijms16010256
OPEN ACCESS
International Journal of
Molecular Sciences
ISSN 1422-0067
www.mdpi.com/journal/ijms
Review

Molecular Targets of Antihypertensive Peptides:

Understanding the Mechanisms of Action Based on the
Pathophysiology of Hypertension
Kaustav Majumder 1,2 and Jianping Wu 1,2,*

1
Department of Agricultural, Food and Nutritional Science, Faculty of Agricultural,
Life and Environmental Sciences, University of Alberta, Edmonton, AB T6G 2P5, Canada;
E-Mail: kaustav@ualberta.ca
2
Cardiovascular Research Centre, Faculty of Medicine & Dentistry, University of Alberta,
Edmonton, AB T6G 2S2, Canada

* Author to whom correspondence should be addressed; E-Mail: jianping.wu@ualberta.ca;

Tel.: +1-780-492-6885; Fax: +1-780-492-4265.

Received: 24 September 2014 / Accepted: 15 December 2014 / Published: 24 December 2014

Abstract: There is growing interest in using functional foods or nutraceuticals for the
prevention and treatment of hypertension or high blood pressure. Although numerous
preventive and therapeutic pharmacological interventions are available on the market,
unfortunately, many patients still suffer from poorly controlled hypertension. Furthermore,
most pharmacological drugs, such as inhibitors of angiotensin-I converting enzyme (ACE),
are often associated with significant adverse effects. Many bioactive food compounds
have been characterized over the past decades that may contribute to the management of
hypertension; for example, bioactive peptides derived from various food proteins with
antihypertensive properties have gained a great deal of attention. Some of these peptides
have exhibited potent in vivo antihypertensive activity in both animal models and human
clinical trials. This review provides an overview about the complex pathophysiology of
hypertension and demonstrates the potential roles of food derived bioactive peptides
as viable interventions targeting specific pathways involved in this disease process.
This review offers a comprehensive guide for understanding and utilizing the molecular
mechanisms of antihypertensive actions of food protein derived peptides.

Keywords: antihypertensive peptides; spontaneously hypertensive rats; hypertension;

angiotensin converting enzyme; endothelin; nitric oxide
Int. J. Mol. Sci. 2015, 16 257

1. Introduction

Cardiovascular diseases account for approximately one third of the total deaths, totaling ~17 million
annually worldwide [1]. Hypertension, the persistent elevation of blood pressure over 140/90 mm Hg
(systolic/diastolic blood pressure, respectively), is considered one of the key risk factors for the
development of cardiovascular diseases (CVD). Hypertension is often termed as “silent killer” affecting
1 billion people worldwide, and causes up to 9 million deaths every year [1]. In Canada, almost 6 million
people (about 1 in every 5 adults), are affected by this condition [2]. Hypertension rarely presents with
early symptoms, and even if diagnosed early, it is often treated inadequately [3,4]. Nevertheless,
hypertension is a significant risk factor for atherosclerosis and hence predisposes to coronary heart
disease, cerebrovascular disease, and renal disease [5–9]. In addition to tremendous health burden,
treatment and prevention of hypertension are also associated with substantial socioeconomic consequences.
The estimated costs for treating hypertension and related diseases were $156 billion in the USA
in 2011 [10]. Pharmacological anti-hypertensive drugs are often associated with significant adverse
side effects such as headache, dry cough, etc. [11,12]. Consequently, many patients still have their
blood pressure poorly controlled and remain at increased risk for its complications even when treated
with existing drugs [13,14]. Therefore, novel, cost-effective and efficient therapeutic strategies are
urgently required for better management of hypertension.
It is well recognized that diet plays an important role in human health. Epidemiological studies have
suggested that food habit or dietary choice can affect the prevalence of chronic diseases such as
cardiovascular disease, obesity, and diabetes [15–17]. Diet manipulation studies such as dietary approaches
to stop hypertension (DASH) suggest that adoption of a healthy diet (rich in fruits and vegetables)
could lower high blood pressure [18,19]. Similarly, compounds like dietary sodium (present in table
salt) and dietary potassium also have a great impact on blood pressure and associated vascular
diseases [20–22]. Moreover, various clinical studies have demonstrated that macronutrients (protein,
fat, and carbohydrate) can play key role in the management of high blood pressure. The optimal
macronutrient intake to prevent heart disease (OmniHeart) trials demonstrated that partial replacement
of carbohydrate with either protein or with monounsaturated fat could reduce high blood pressure, and
the risk of coronary heart disease [23–25]. Indeed, food proteins also contain active peptide fragments
encrypted within their structure that can exert beneficial effects on human health above and beyond
their expected nutritional value. These active peptide fragments, known as bioactive peptides, can be
released from their parent proteins by gastrointestinal digestion, fermentation, or food processing [26].
Food derived bioactive peptides have vast potential for applications as functional foods and nutraceuticals
for the prevention and management of hypertension.
Among many types of food derived bioactive peptides, peptides with antihypertensive activity have
received the most significant attention due to the persistence of hypertension and its associated
complications even with pharmacological interventions [27–29]. These peptides target mainly at inhibiting
angiotensin I converting enzyme (ACE), an enzyme playing a crucial role through renin angiotensin
system (RAS) for the regulation of blood pressure and electrolyte balance in human body [7,30,31].
Peptides with anti-oxidant, anti-inflammatory, opioid receptor binding activities might also exhibit
anti-hypertensive activity [32,33]. However correlation between in vitro and in vivo antihypertensive
activities appears to be weak [29,32,34–38]. To develop effective antihypertensive peptides, it is important
Int. J. Mol. Sci. 2015, 16 258

to understand the complex pathophysiology of hypertension and the potential targets where these bioactive
peptides may exert their specific antihypertensive actions. The potential mechanisms of action of many
food-derived peptides with antihypertensive activity have been previously reviewed [28,29,39–42].
However, limited information is available regarding the multiple functional roles of these peptides on
various pathways involved in developing persistent hypertension.
Therefore, this particular review provides an overview about the complex pathophysiology of
hypertension and highlights potential molecular targets of food derived peptides that may mediate the
in vivo antihypertensive effects. Identification of these molecular targets can facilitate the use of food
derived bioactive peptides as a novel therapeutics for the prevention and management of hypertension.

2. Pathophysiology of Hypertension

Hypertension develops from a complex interaction of genetic and environmental factors although
more than 90% of cases do not have a clear etiology [43,44]. Previous research has identified major
contributing factors: (i) increased sympathetic nervous system activity; (ii) increased levels of long term
high sodium intake, inadequate dietary intake of potassium and calcium; (iii) altered renin secretion
related to elevated activity of the RAS; (iv) increased activity of ACE resulting over production of
angiotensin II (Ang II) and deactivation of kallikrein kinin-system (KKS); (v) endothelial dysfunctions
and deficiencies of vasodilators including reduced nitric oxide (NO) bioavailability; (vi) abnormalities
in vessel resistance due to vascular inflammation, increased activity of vascular growth factors and
altered cellular ion channel [45–49]. Although all of the above factors clearly contribute to the pathogenesis
of hypertension, the hyperactivity of the RAS, endothelial dysfunction, enhanced activation of sympathetic
nervous system and structural abnormalities in resistance vessels play critical roles in the development
and progression of this disease [49–51].

2.1. Renin Angiotensin System (RAS)

Physiologically, RAS is one of the important pathways for regulating blood pressure and vascular
tone in human body [52,53]. The RAS pathway is initiated in the kidney with the proteolytic conversion
of angiotensinogen to angiotensin I (Ang I) by renin. Ang I is an inactive decapeptide which can be
converted into a vasoconstrictive octapeptide, Ang II, by the action of ACE. Ang II can be further
cleaved by angiotensin converting enzyme 2 (ACE-2), to form angiotensin 1–7 (Ang1–7), then the
G-protein-coupled receptor (GPCR)-Mas acts as an Ang1–7 receptor and initiates a counter-regulatory
role by opposing Ang II induced vasoconstriction [7,54]. In addition, ACE-2 can also cleave a single
amino acid from Ang I, producing inactive angiotensin 1–9 (Ang1–9). Ang II is an important regulator
of fluid and sodium balance and also participates in cellular growth and remodeling [52,55].
Ang II acts through two main receptors, angiotensin type 1 (AT1) and type 2 (AT2) receptors [30,53]
(Figure 1). Binding to AT1 receptor causes vasoconstriction in vascular smooth muscle cells
(VSMC). It also stimulates release of aldosterone to increase water and salt retention in the kidney,
hypertropic growth of cardiomyocytes, and collagen synthesis of cardiac fibroblasts resulting in
cardiac remodeling. In pathogenic conditions involving tissue remodeling and vascular inflammation,
AT1 receptor is up regulated [56–58]. On the other hand, AT2 receptor presents in both endothelial
and VSMC mediates vasodilation upon activation, releases NO, and inhibits cell growth [59]. Therefore,
Int. J. Mol. Sci. 2015, 16 259

AT1 receptor mediates actions with potentially harmful consequences, whereas AT2 receptor, mediated
actions exhibits protective effects against hypertension [53,60] (Figure 1).
Alternatively, ACE also actively participates in the KKS. Activation of ACE inactivates bradykinin,
a potent vasodilator. Bradykinin acts through two different receptors, type 1 (B1) and type 2 (B2).
Both receptors induce NO generation in endothelial cells [61,62]. In addition, B2 receptors also activate
phospholipase A2 that releases arachidonic acid, which leads to the formation of several vasodilators
including prostacyclin [30,31,53,63].
Though RAS is widespread in the body, the main source of renin is the juxtaglomerular apparatus
of the kidney, while that of ACE is abundantly present cell surface of endothelial cells, especially in
the lungs [64,65]. However, there is increasing evidence supporting an important role of local RAS, such
as in the microvasculature of kidney, heart, and arterial tree, in the regulation of blood pressure [30,55,66].

Figure 1. Renin-angiotensin system and kallikrein kinin system to regulate of blood pressure.
Angiotensin I (Ang I), Angiotensin II (Ang II), Angiotensin converting enzyme (ACE),
Angiotensin converting enzyme 2 (ACE 2), Angiotensin receptor 1 (AT1), Angiotensin
receptor 2 (AT2), Bradykinin receptor 1 (B1), Bradykinin receptor 2 (B2), Nitric oxide (NO),
Prostaglandins 2 (PgI2). Figure 1 modified from [63].

2.2. Endothelial Dysfunction

Endothelial cells (EC) play important physiological functions in regulation of the vascular homeostasis
or vascular balance under normal conditions. Impairment of normal vasorelaxant EC responses results
in endothelium dysfunction. Endothelial dysfunction often disturbs the vascular function and creates
Int. J. Mol. Sci. 2015, 16 260

a vascular imbalance that is responsible for various cardiovascular diseases including hypertension.
Endothelial cells produce a number of vasoactive substances, including NO and endothelin (ET-1).
NO the key vasodilator, and ET-1 a potent vasoconstrictor, are vital mediators of endothelial functions.
An imbalance between these two factors is a feature of endothelial dysfunction.
NO initiates and maintains vasodilation through a cascade of biological events after diffusing through
cell membrane [59]. NO is generated in endothelial cells by nitric oxide synthase (NOS) in a two-step
five-electron oxidation of the terminal guanidine nitrogen of L-arginine, generating L-citrulline as
a by-product. Three isoforms of NOS have been characterized: endothelial NOS (eNOS), neuronal NOS
(nNOS) and inducible NOS (iNOS) [67]. Both eNOS and nNOS are present in the normal vascular
endothelium [68,69]. After diffusion from endothelial to vascular smooth muscle cells, NO causes
vasodilation [69] primarily by activating soluble guanylyl cyclase (sGC) and increasing intracellular
concentration of cyclic guanosine-monophosphate [70] (Figure 2). Acute NOS inhibition results in
vasoconstriction and reduction in peripheral blood flow [64]. These hemodynamic alterations are
entirely reversible with administration of NO donors, such as glyceryl trinitrate (GTN) or sodium
nitroprusside (SNP) [71], suggesting that the continuous presence of NO is required to prevent
vasoconstriction. In addition, NO also affects cell metabolism, and inhibits mitochondrial respiration
and ATP synthesis [59].
It has been suggested that NO bioavailability can be reduced in the presence of excessive reactive
oxygen species (ROS) such as superoxide anion (O2−). Ang II enhances the formation of superoxide
in endothelial cells by activating nicotinamide adenine dinucleotide phosphate (NADPH) oxidase.
Superoxide readily reacts with NO to form peroxynitrite (ONOO−). Peroxynitrite is a strong oxidant,
causing damage to cell membrane while leads to cell death and/or inflammation [72] (Figure 2).
Excessive formation of O2− also modifies tetrahydrobiopterin (BH4) a cofactor for NOS [64]. In the
absence of this cofactor, NOS can become uncoupled and paradoxically generated O2− instead of
NO [58]. Decreased bioactivity of NO could switch the cellular signaling from NO-mediated cellular
processes to oxidant-mediated redox signaling, stimulating pro-inflammatory pathways, and ultimately
leading to vascular remodeling and resulting in increased blood pressure [73].
In contrast to NO, circulating endothelins have vasoconstrictory properties. Three isoforms for
endothelins (ET-1, ET-2, and ET-3) have been characterized but ET-1 is the dominant form and actively
modulates vascular functions [74]. ET-1 is synthesized predominantly in endothelial cells and also in
vascular smooth muscle cells [74]. Its precursor, preproET-1 (ppET-1) is a functionally inactive peptide
which is sequentially cleaved by cellular enzymes and ultimately produces the vasoactive ET-1.
Furin-like proteases cleave ppET-1 to generate a 39 amino acid peptide (38 amino acids in humans)
called big-ET-1 (bET-1). Under normal physiological conditions, endothelin-converting enzyme (ECE)
converts big endothelin (bET-1) to ET-1, whereas current evidence suggests ET-1 can be produced
from bET-1 through several other proteolytic digestions involving matrix metalloproteinases (MMPs),
and neutral endopeptidase (NEP). ET-1 exerts its functions by binding to G protein-coupled ET receptors,
endothelin receptor A (ETA) and endothelin receptor B [75]. ETA receptors are located within the VSMC,
whereas ETB receptors are located both on vascular endothelium, as well as, on VSMC. Binding with
ETA and ETB receptors in vascular smooth muscle ET-1 exerts vasoconstriction. Alternatively, ET-1
binding to ETB receptors in the endothelium results in vasodilation through increased NO and prostacyclin
synthesis [74]. The interplay between NO and ET-1 is important in numerous pathophysiological
Int. J. Mol. Sci. 2015, 16 261

conditions [76]. The reduction in NO bioavailability is associated with increased ET-1 expression.
Similarly, NO antagonizes the ET-1 pathway via several different mechanisms [75]. These relationships
suggest an intimate link between these two mediators to maintain a delicate balance in endothelial
function [74]. ET-1 also stimulates the release of pro-inflammatory cytokine such as interleukin (IL)-1,
and IL-8. Factors like Ang II, thrombin, and inflammatory cytokines (tumor necrosis factor-α, IL-1,
IL-2) can modulate the expression of ET-1 in endothelial cells by enhancing the gene expression of
ppET-1. Therefore reduced bioavailability of NO and excessive production of Ang II can directly induce
endothelial dysfunction and subsequent increase in blood pressure.

Figure 2. Endothelial dysfunction and blood pressure regulation. Angiotensin converting

enzyme (ACE) converts angiotensin I (Ang I) to angiotensin II (Ang II), Ang II binds with
angiotensin receptor 1 (AT1) on endothelium cells as well as vascular smooth muscle cells,
then AT1 receptor increases calcium ion (Ca2+) concentration in vascular smooth muscle
cells (VSMC) and exerts vasoconstriction. In endothelium cells activation of AT1 receptor
increases the production of bET-1 (big endothelin-1). Endothelin-Converting Enzyme (ECE)
converts bET-1 to endothelin-1 (ET-1) and exerts vasoconstriction by activating endothelin
A/B receptors (ETA/B) in the VSMC. In contrast, activation of ETB receptor in endothelium
cells mediates vasodilatory effects via release of nitric oxide (NO) by activating endothelial
nitric oxide synthase (eNOS). ACE also converts Bradykinin (Bk) into inactive peptides.
Bk binds with bradykinin receptor (B1/2) and activates eNOS, which converts L-Arginine to
L-Citrulline and produces NO. NO exerts vasodilation by activating cyclic guanosine
monophosphate (cGMP) by inhibiting the concentration of Ca2+ in VSM. In endothelium
cells Ang II produces superoxide (O2−) which scavenges NO and produces peroxynitrite
(ONOO−), exerts vasoconstriction effect by limiting the supply of NO to the VSM. Signaling
pathways illustrated with solid line arrows are representing vasoconstriction and with
compound line arrows are representation vasodilation network. Figure 2 modified from [71].
Int. J. Mol. Sci. 2015, 16 262

2.3. Sympathetic Nervous System

The sympathetic nervous system is a part of the autonomic nervous response system that can be
activated by environmental stress. Increased sympathetic nervous system activity can cause both arteriolar
constriction and arteriolar dilation [77]. Thus, the autonomous nervous system contributes to the
development and maintenance of hypertension through stimulation of cardiac output in heart, fluid
retention in kidney and increased vascular resistance in peripheral vasculature [77].
Sympathetic nervous system stimulates the release of catecholamines (norepinephrine and epinephrine)
from postganglionic neurons [78]. The release of catecholamines activates the hypertrophic growth of
cardiomyocytes [79]. Simultaneously, catecholamine release increases the activity of β-adrenoceptors
while decreases the activity of α-adrenoceptors, which in turn results in the conversion of pro-renin to
the active form of renin [79]. The release of renin subsequently activates RAS and results in increased
blood pressure through the production of Ang II (Figure 3). Ang IAng II also amplifies the response of
the sympathetic nervous system by a peripheral mechanism, that is, pre-synaptic facilitatory modulation
of norepinephrine release [77,78]. Additionally, ROS and ET-1 may also stimulate the sympathetic
activity and its effects on the vasculature [78,80]. Thus increased sympathetic activity is associated
with the development of hypertension.

Sympathetic Nervous System

Cathecholamines

ET-1 ROS
Hypertrophic Renin release
growth of
cardiomyocytes

α1-adrenergic RAS activity Ang II

receptors

Vasoconstriction Vasoconstriction

Figure 3. Regulation of blood pressure through autonomic nervous system. Increased

sympathetic nervous system stimulates the release of cathecholamines from post ganglionic
neurons. Cathecholamines increases the hypertrophic growth of cardiomyocytes and release
more renin in adrenal cortex. Increase production renin over activates renin angiotensin system
(RAS) and produces more Angiotensin-II (Ang II). Hypertrophic growth of cardiomyocytes
and increase production of Ang II results in vasoconstriction. In addition, Ang II production
increases ET-1 (Endothelin-1) and ROS (reactive oxygen species) production and directly
affect the over activity of sympathetic nervous system.
Int. J. Mol. Sci. 2015, 16 263

2.4. Vascular Remodeling

Vascular remodeling contributes to increased peripheral resistance, alterations in vessel structures,

development of hypertension, and the consequent end organ damage during hypertension [6,48].
Hypertension associated with structural changes in the vessels has been called as remodeling. Vascular
remodeling is an active process and involves changes in cellular processes, such as cell growth,
cell migration, cell death, and degradation/synthesis of extracellular matrix [48,81]. Remodeling of
vessels increases peripheral resistance in pre-capillary vessels including arterioles and small arteries
(lumen diameters < 300 µm). These structural changes of the vessels reduce the lumen diameter and
increase the media-to-lumen ratio (M/L) and ultimately, increase both vascular reactivity and peripheral
resistance [48].
Vascular inflammation can induce endothelial dysfunction, which ultimately results in vascular
remodeling. Under resting conditions, EC prevent leukocyte adhesion but local overproduction of
Ang II can initiate the expression of adhesion molecules on endothelial cells which results in adhesion
of leukocyte to the inner arterial wall in a stepwise manner, known as leukocyte recruitment [82].
Subsequently Ang II can also induce oxidative stress resulting in excessive production of ROS. ROS,
in turn, stimulates the production of cytokines such as tumor necrosis factor-α (TNF-α), IL-1β etc. [83].
TNF-α activates the phosphorylation of nuclear transcription factor-κB (NF-κB), which leads to
the expression of adhesion molecules (ICAM-1, intercellular adhesion molecule-1; VCAM-1,
vascular cell adhesion molecule-1), and the release of monocyte chemotactic protein-1 (MCP-1).
The over-expression, of these molecules then recruits the leukocytes (monocytes and macrophages)
to the site of inflammation [82]. Over-expressed inflammatory response together with the oxidized
lipid molecules forms plaques in the interstitial space between endothelial and vascular smooth
muscle. Formation of plaque directly contributes to vascular remodeling, increases blood pressure and
initiates atherosclerosis.
Matrix metalloproteinases (MMP) are zinc (Zn) and calcium (Ca) dependent proteolytic enzymes
that degrade extracellular matrix proteins [84]. Several different MMPs are present in the vasculature
and their synthesis is induced by cytokines and cell to cell-matrix interactions. An increasing body of
scientific evidence demonstrates that uncontrolled proteolytic process is one of the key mechanisms for
the development of hypertension and MMP play a crucial role in this process [84,85]. In Ang II-induced
hypertension, MMP are responsible for elevated blood pressure and tissue fibrosis [85]. Acute release
of MMP2 cleaves the sarcomere proteins (titin, troponin I and myosin light chain-I) that can impair
cardiomyocyte contractility [86,87]. Similarly, MMP-7 is one of the major inducing factors for endothelial
dysfunction. MMP-7 also promotes GPCR agonist (i.e., Ang II) induced vasoconstriction through
epidermal growth factors (EGF) and subsequently increases the blood pressure and cardiovascular
hypertrophy [88]. Thus MMP with accessory signaling molecules can modulate cell-cell interaction,
release of cytokine, and chemokines, which ultimately propagate the vascular inflammatory response.
Apart from the pathways described above, there are other factors such as sodium/water excretion,
adrenal steroids, etc. that also contribute to the development of high blood pressure. Interestingly,
one or more component of each distinctive pathway can modulate or activate another pathway and
thus create a complex cycle for the development of hypertension (Figure 4). The pathophysiologic
mechanisms that lead to blood pressure elevation are so complex that anti-hypertensive treatment
Int. J. Mol. Sci. 2015, 16 264

based on a single pathway is rarely feasible (ACE inhibitors may be an exception, although Ang II
involves both RAS and pro-inflammatory pathways); [89]. Current pharmacological treatment approaches
for treating hypertension are very much selective, which may explain the inadequacy in palliation of
hypertension and adverse side effects. Food protein-derived peptides have been widely studied for
controlling elevated blood pressure, but it is essential to understand their effects on the pathogenic
mechanisms and the interplay between different molecules to develop these as novel therapeutic agents.

Renin angiotensin system

AGT Ang I Ang II
Renin

Endothelial dysfunction
Sympathetic nervous
system
ET-1 NAD(P)HOx NF-κB AT1

O2- NO

Up-regulation of inflammatory molecules VASOCONSTRICTION

ICAM1, VCAM-1, TNF-α, IL-1β etc

Vascular remodeling

Figure 4. Pathophysiology of hypertension—a vicious cycle. Renin angiotensin system

(RAS), endothelial dysfunction, vascular remodeling, and activity of sympathetic nervous
system are correlated with each other. Enhanced RAS activity leads to over production of
angiotensin II (Ang II) which accelerates endothelial dysfunction. Ang II induced endothelial
dysfunction results in vasoconstriction as well up regulates the activity of transcription
factors (such as NF-κB, nuclear factor κB), promoting vascular inflammation. Vascular
inflammation up regulates the expression of leukocyte adhesion molecules such as
ICAM-1 (Intercellular adhesion molecule 1), VCAM-1 (Vascular adhesion molecule-1)
as well as inflammatory cytokines like TNF-α (Tumor necrosis factor-α) and IL-1β
(Interleukin-1β). Similarly, during endothelial dysfunction over expression of ET-1
(Endothelin-1) and increased levels of ROS such as superoxide (O2−) can directly increase
the sympathetic nervous system. Finally, increased sympathetic nervous system increases
renin production which eventually activates RAS.

3. Antihypertensive Peptides from Food Proteins—Mechanisms of Action

Food protein-derived peptides exhibit antihypertensive effects through various mechanisms. A majority
of food protein-derived antihypertensive peptides have been initially identified as ACE inhibitors
using in vitro methods. Peptides with ACE inhibitory properties were isolated first from snake
(Bothrops jararaca) venom [90,91]. This work encouraged several subsequent investigations to look
Int. J. Mol. Sci. 2015, 16 265

for food protein derived peptides as antihypertensive alternatives. Earlier studies have identified
several ACE-inhibiting peptides from both plant (especially soybean) and animal sources (milk, fish
and egg proteins) [27,92–95]. Given the complexity of blood pressure regulation, it is important to
understand the mechanism of action of a peptide in order to develop functional foods or nutraceuticals
for the prevention and management of hypertension. The following sub-sections briefly describe
the effect of food-derived bioactive peptides on modulating RAS function, ameliorating endothelial
dysfunction, modulating sympathetic nervous system, and vascular inflammation.

3.1. Antihypertensive Peptides Modulating RAS Function

ACE inhibition is the main mechanism by which peptides can modulate RAS function and exert
antihypertensive effects. A number of in vivo studies performed in animals and/or humans have
demonstrated that various food-derived peptides could significantly reduce blood pressure through
ACE inhibition upon either intravenous or oral administration [93–100]. Milk protein derived peptides
are known for their antihypertensive activity. The release of antihypertensive peptides from milk
protein has been achieved through two different approaches: hydrolysis of milk protein by proteolytic
enzymes and fermentation of milk. One of the first peptides identified from tryptic digestion of
αs1-casein, FFVAPFPGVFGK, could significantly reduce both systolic blood pressure (SBP by
34 mmHg) and plasma ACE activity at a dose of 100 mg/kg BW in spontaneously hypertensive rats [38].
MKP, another peptide identified from the tryptic digest of bovine casein has also shown antihypertensive
effect in SHRs. The crude hydrolysate containing only 0.053% of MKP significantly reduced the SBP
by 40 mmHg at a dose of 100 mg/kg BW 2 h after administration, whereas the purified peptide MKP
exhibited a maximum SBP reduction of 45 mmHg 8 h after administration in SHR. Both preparations
also exhibited ACE inhibitory properties [101]. Three peptides, IAK, YAKPVA, and WQVLPNAVPAK
from αs1-casein produced by combined action of pepsin, chymotrypsin, and trypsin showed a significant
decrease in both SBP and diastolic blood pressure (DBP) in SHR with doses of 4, 6, and 7 mg/kg BW,
respectively [99]. The authors determined that the ACE inhibitory property of these peptides was
responsible for the observed antihypertensive effect [99]. Two tripeptides, VPP and IPP, produced
from milk fermentation with a combination of Lactobacillus helveticus and Saccharomyces cerevisiae,
were the well-known antihypertensive peptides from milk [102,103]. Single oral administration of
VPP and IPP at a dose of 0.6 and 0.3 mg/Kg BW could significantly reduce SBP by 32 and 28 mmHg,
respectively [104]. SHRs fed with fermented milk containing these peptides demonstrate significant
decreases in serum ACE activity and BP [104].
Apart from milk, fish protein derived peptides have also been shown to exhibit antihypertensive
effect through ACE inhibition. Three peptides LKP, IKP, and IWH identified from hydrolysate of
dried bonito have been shown to significantly reduce SBP in SHR animals [29,105]. Another peptide
LKPNM, also identified from bonito hydrolysate, was found to exert a longer-term effect on SHRs
than LKP. The authors identified LKPNM as a “pro-drug” type ACE inhibitor, which could serve as
a precursor to the actual ACE inhibitor released upon gastrointestinal proteolysis [105].
Egg protein ovalbumin derived peptide YPI reduces blood pressure by 30 mm Hg after a single oral
administration to SHRs and its actions were likely mediated through ACE inhibition [94]. A study
from our own research group identified a potent ACE inhibitory tri-peptide IRW from thermolysin-pepsin
Int. J. Mol. Sci. 2015, 16 266

hydrolysate of egg white protein ovotransferrin [106]. In SHRs, IRW significantly reduced SBP by
40 mmHg after 18 days of treatment at a dose of 15 mg/kg BW while concomitantly decreased plasma
Ang II levels, likely through ACE inhibition [97].
AT1, the Ang II receptor, is one of the targets to modulate increased RAS activity. In addition
to inhibition of ACE, AT1 receptor blockade is considered as an effective therapy for hypertensive
patients [107]. Moreover, it is a useful alternative approach for the patients sensitive to side effects of
ACE inhibitors [107]. Similarly, renin is a key regulator of RAS; therefore inhibition of renin could
also alter RAS activation. Milk lactoferrin derived peptides RRWQWR, LIWKL, and RPYL significantly
reduced blood pressure in SHRs and were also found to reduce Ang II induced vasoconstriction in
isolated rabbit carotid arterial segments [108]. Among these three peptides, RPYL showed the maximum
effect, and demonstrated inhibition of Ang II binding to AT1 receptors [109]. Recently, egg protein
derived peptide RVPSL has been shown to significantly decrease SBP by 25 mmHg after 4 weeks of
treatment at a dose of 50 mg/Kg BW. The mRNA levels of renin, ACE, and AT1 receptor in kidney
and serum level of AngII and renin were all significantly decreased by RVPSL treatment [109].
Results from these studies suggest that food derived bioactive peptides can indeed act upon the AT1
receptor and/or act as a renin or ACE inhibitors to exert their in vivo antihypertensive effects [109].
Likewise, renin inhibition is also a crucial mechanism of controlling blood pressure by reducing
the formation of Ang II. Glycyl histidinyl serine (GHS), a peptide isolated from pepsin, pancreatin
digestion of rapeseed protein exhibited both ACE and renin inhibitory activity and oral administration
of this peptide (30 mg/kg BW) reduces blood pressure in SHR 6 h after administration [110].
Interestingly in a different study, Ehlers et al. had demonstrated that the vasorelaxation effect of
IPP may be mediated though ACE-2, Ang1–7, and Mas axis. In an ex vivo experiment the authors
demonstrated that the administration of IPP can produce more Ang1–7 and exert vasorelaxation activity
on Mas receptor possibly through modulation of ACE-2 [111]. Similarly, study from our group
has found that IRW treatment could increase the gene expression of ACE-2 in mesenteric artery
(unpublished data), which may further convert Ang II to Ang1–7 and exerts vasodilation. Thus activation
of ACE-2 through peptide treatment could exert beneficial effect for the prevention of hypertension.
Peptides previously identified as ACE inhibitors though in vitro method could actually exhibit various
effects on the RAS and thus reduce blood pressure.

3.2. Antihypertensive Peptides Ameliorating Endothelial Dysfunction

Increased production of vasoconstrictory/pro-inflammatory mediators like ET-1 and superoxide

(O2−) decreases the bioactivity of vasodilatory NO resulting in endothelial dysfunction. Food protein
derived antihypertensive peptides have been shown to improve endothelial functions and cause
vasodilation. The three main mechanisms by which food derived antihypertensive peptides modulate
endothelial function are increased production of vasodilatory factors (i.e., NO and prostaglandins),
reduced production of vasoconstriction factors (i.e., ET-1), and increased anti-oxidant activity.
Egg protein ovalbumin derived peptide RADHP could significantly reduce blood pressure by 28 mmHg
after a single oral administration in SHR animals [112]. RADHP also exhibited a dose-dependent
relaxation in an isolated SHR mesenteric artery. However, the removal of endothelium from mesenteric
artery was associated with disappearance of the relaxation effect, suggesting endothelium-dependent
Int. J. Mol. Sci. 2015, 16 267

vasodilator activity of RADHP [113]. Moreover, pretreatment with N-nitro-L-arginine methyl ester
(L-NAME), a NOS inhibitor, inhibited the RADHP mediated vasodilation; while pretreatment with
superoxide dismutase (SOD), a radical scavenger, did not alter RADHPF induced vasodilation, suggesting
that the vasodilatiory effect was unlikely to be caused by scavenging (O2−), but possibly resultant from
stimulating NO production [114]. Four other peptides, YRGGLEPI, YR, ESI, and NF from egg protein
ovalbumin have also demonstrated in vivo antihypertensive effect in SHRs [94,115]; their actual
mechanisms of action remain unknown. However, it was evident that these antihypertensive effects
were independent of ACE inhibition, as in an isolated mesenteric artery experiment vasorelaxation
activity of these peptides were blocked by the treatment of NO inhibitor L-NAME and cyclooxygenase
inhibitor indomethacin [29,115]. In addition to their ACE inhibitory effects, VPP and IPP also
demonstrate the capability to improve vascular endothelial dysfunction. Yamaguchi et al. demonstrated
that VPP and IPP administration could significantly increase the mRNA expression of eNOS in
SHR [115]. Increased expression of eNOS directly correlates with enhanced production of NO and
reduction of BP. A later study also showed that administration of VPP and IPP to cultured endothelial
cells could significantly increase the NO production [116–118]. Results from these studies clearly
indicate that antihypertensive peptides VPP and IPP could induce vasodilation through NO,
independently of ACE inhibition. Results from our previous study also demonstrate that egg protein
ovotransferrin derived peptide IRW treatment could increase the NO mediated vasodilation in
mesenteric arteries of SHR animals, probably through increasing eNOS expression [97].
Increased bioavailability of NO can also improve vasodilation and reduce BP. Therefore, antioxidant
and free radical scavenging activities could be beneficial for altering endothelial dysfunction. Milk-derived
peptides RYLGY and AYFYPEL, obtained from bovine casein hydrolysate, have shown in vivo ACE
inhibitory and anti-oxidant effects [118]. Oral administration of these peptides significantly reduced BP
in SHR animals at a dosage of 5 mg/kg BW. The authors conclude that in vitro ACE inhibitory activity
and radical scavenging activity of these peptides could potentially contribute towards reduction in
blood pressure in SHR [118]. Therefore, the vasoprotective activity of these peptides could reduce
blood pressure and potentially ameliorate the vascular fibrosis. Additionally, another peptide MY, derived
from sardine muscle, exhibited antihypertensive effect by suppressing ROS generation in endothelial
cells via induction of hemeoxygenase-1 (HO-1) and ferritin [119]. A study from our group has shown
that the egg derived peptides IRW and IQW could significantly reduce TNF-α induced oxidative stress
in cultured endothelial cells [120]. Moreover, oral administration of IRW could reduce oxidative stress
in aorta and kidneys in intact SHR animals [97]. Thus, by acting as an anti-oxidant, these peptides play
a crucial role to improve NO bioavailability and consequently modulate endothelial function and BP.
Apart from increased bioavailability of NO, treatment of bioactive peptide can also release various
other vasodilatory factors such as prostaglandins (PGI2). Zhao et al. identified an antihypertensive peptide
MRW from pepsin-pancreatin digest of spinach Rubisco (Ribulose bisphosphate carboxylase/oxygenase)
protein. Oral administration of MRW at a dose of 5 mg/Kg BW could significantly reduce the SBP by
20 mmHg in 25 week old male SHR [121]. MRW also exhibited a dose-dependent vasodilation in
an ex vivo study on isolated mesenteric arteries of SHRs; the relaxation effect of MRW was not
NO-dependent, and was mediated by upregulation of PGI2 possibly through B2 receptor activation [121].
A similar effect was observed with RIY, a peptide derived from the rapeseed protein napin [122].
Int. J. Mol. Sci. 2015, 16 268

The study by Yamada et al. suggested that the antihypertensive effect of RIY in SHRs is induced
mainly by the production of PGI2 [123].
The interplay between NO and ET-1 is well established in the context of endothelial dysfunction.
The ECE plays an important role in converting inactive bET-1 to vasoactive ET-1, which subsequently
binds to ET receptors and induces vasoconstriction. Therefore, ECE inhibitors or ET receptor agonists
are the key targets for the antihypertensive therapy [124]. Bovine β-lactoglobulin derived peptide
ALPMHIR was found to suppress the ET-1 activity in porcine aortic endothelial cells [125], possibly
through ECE inhibition. In another study, eight peptides derived from lactoferricin B those
were previously characterized as ACE inhibitors have showed significant inhibition of ECE
activity when vasoconstriction was induced by big ET-1. Lfcin17–25 (FKCRRWQWR), LfcinB17–31
(FKCRRWQWRMKKLGA), LfcinB17–32 (FKCRRWQWRMKKLGAP), and Lfcin19–25 (CRRWQWR),
were the most potent among them; these peptides were shown to inhibit ECE in isolated aortic
segments from rabbits [126]. Furthermore, in a follow up study, the same group identified two
more ECE inhibitory peptides, GILRPY and REPYFGY, from bovine lactoferrin hydrolysate [127].
Interestingly, these studies also suggested that these peptides may act either as dual vasopeptidase
inhibitors (ACE/ECE), or as specific ECE inhibitors to produce their vasorelaxant effects [127]. So far
there is little evidence to prove the relationship between ACE and ECE inhibition [127]; therefore
these peptides may have dual enzyme inhibitory effect, which might result in pronounced blood pressure
reducing effects.
Endothelial dysfunction leads to influx of calcium ion (Ca2+) in VSMC and increases vasoconstriction
by activation of AT1 and ETA/ETB receptors (Figure 2). Therefore, blocking of calcium channel reduces
influx of Ca2+ and results in vasodilation. Peptides derived from fish protein hydrolysate have been
shown antihypertensive effects by blocking the Ca2+ channels [128]. VY, a peptide derived from sardine
muscle, exhibited an antihypertensive effect in SHRs as well as in Tsukuba-Hypertensive Mouse
(THM) at doses of 10 and 0.1 mg/g BW respectively [129,130]. A study by Tanaka et al. showed that
the antihypertensive effect of VY is actually mediated upon the VSM and it acts as an L-type Ca2+
channel blocker [128]. Similar mechanism has also been proposed for the sardine-derived peptide WH
that suppresses the extracellular Ca2+ influx by blocking the L-type Ca2+ channel blocker in human
VSM cells [131,132].

3.3. Antihypertensive Peptides Modulating Sympathetic Nervous System and Controlling

Blood Pressure

Opioid receptors are present in the central nervous system and they are involved in the regulation of
blood pressure through increasing the activity of the sympathetic nervous system [133]. A peptide
(YGLF) derived from pepsin/trypsin digestion of α-lactorphin has been shown to reduce blood pressure
in SHR by binding to opioid receptors [134]. The vasodilatory effect of YGLF is endothelial dependent
and can be inhibited by selected eNOS inhibitors [135]. Therefore, a novel mechanism of binding to
endothelial opioid receptors and subsequent NO release might be responsible for the vasodilatory
effects of this peptide.
Int. J. Mol. Sci. 2015, 16 269

3.4. Antihypertensive Peptides Modulating Vascular Remodeling

Vascular inflammation-induced peripheral resistance is a contributor to elevated blood pressure and

associated vascular pathologies [81]. Milk-derived tripeptide VPP pretreatment could significantly decrease
phorbol 12-myristate 13-acetate (PMA) induced monocyte adhesion to human endothelial cells [136].
In addition, treatment with the both VPP and IPP offered protection against the development of
atherosclerotic plaques in the apolipoprotein E (ApoE) knockout mice through a combined action
involving the modulation of inflammatory as well as hypertensive pathways [137]. In our laboratory,
the egg-derived tri-peptide IRW has demonstrated antihypertensive and anti-inflammatory properties
by controlling both the hyperactive RAS pathway as well as the inflated pro-inflammatory cytokine
levels in SHR [97]. Increased circulating levels of proinflammatory cytokines during the hypertension
could further contribute to endothelial dysfunction and up-regulation of leukocyte adhesion in the
vasculature, which ultimately results in vascular remodeling. Therefore, peptides that control the
inflammatory pathways could potentially improve the vascular pathogenesis, and hence, control vascular
remodeling and reduce elevated blood pressure.
To date, several food protein-derived antihypertensive peptides have been reported with a significant
antihypertensive activity in animal studies, mostly with SHR (Table 1). Based on current available
scientific evidences, it can be concluded that food protein-derived peptides may exert antihypertensive
activity through multiple mechanistic pathways as follows: ACE inhibition, renin inhibition, ACE-2
activation, AT1 receptor blocking, increase NO production, ECE inhibition, PGI2 activation, blocking
of Ca2+ channel, opioid activity, anti-oxidant activity and anti-inflammatory activity (Figure 5).
Different peptide sequences have different modes of action, which may be mediated through their
structural features. Various vasodilatory mechanisms of different peptides are summarized in Table 1.

Bradykinin
(B1) receptor
eNOS up regulation activation

ACE 2 up regulation Increased NO production

Angiotensin ECE Inhibition

receptor
blocking
Antihypertensive effect Calcium
Renin Inhibition channel
blocking

Prostaglandin release
ACE Inhibition
Opioid
agonist
Anti-oxidant activity activity Anti-inflammatory activity

Figure 5. A schematic diagram of antihypertensive mechanism of food derived peptides.

ACE (Angiotensin-I converting enzyme), ACE 2 (Angiotensin converting enzyme 2), eNOS
(Endothelial nitric oxide synthase), NO (Nitric oxide), ECE (Endothelin converting enzyme).
Int. J. Mol. Sci. 2015, 16 270

Table 1. Antihypertensive activity and vasodilatory mechanism of food derived bioactive

peptides in spontaneously hypertensive rats.
Vasodilatory Peptide SBP Decrease
Food Protein Dose mg/kg BW References
Mechanism Sequence (mm Hg)
ACE inhibition Milk α-casein MKP 0.5 −30.0 [101]
κ-casein IAK 4.0 −20.7 [138]
YAKPVA 6.0 −23.1 [139]
β-casein IPP 0.3 −28.3 [102,117]
VPP 0.6 −32.1 [102,117]
Egg Ovotransferrin IRW 15.0 −40.0 [97,106]
Fish Bonito muscle LKP 2.25 −5.0 [105]
AT1 blocker Egg Egg white protein RVPSL 50.0 −25.0 [109]
2+
Ca channel
Fish Sardine Muscle VY 10.0 −18.5 [129]
blocker
PGI2 activator Rapseed Napin RIY 7.5 −28.0 [123]
Spinach Rubisco MRW 5.0 −20.0 [121]
Renin inhibition Egg Egg white protein RVPSL 50.0 −25.0 [109]
ACE-2
Milk β-casein IPP 0.3 −28.3 [111]
activation
Anti-oxidant Milk α-casein MKP 0.5 −30.0 [102]
RYLGY 5.0 −32.0 [139]
MY 10.0 −19.4 [39,119]
Opioid-agonist Milk α-lactorphin YGLF 1.0 −23.7 [134]
eNOS
Milk β-casein IPP 0.3 −28.3 [115]
up-regulation
VPP 0.6 −32.1 [115]
Egg Ovotransferrin IRW 15.0 −40.0 [97,106]

4. Antihypertensive Effects of Food Derived Peptides—Clinical Studies

Clinical trials are necessary to evaluate the efficacy of food protein derived bioactive peptides in
humans. It is also important to study the pharmacokinetics for the development of nutraceuticals and/or
functional foods from food protein derived bioactive antihypertensive peptides. Two well-known
peptides, VPP and IPP have shown efficacy as antihypertensive agents in human clinical studies [139].
Oral administration of VPP and IPP incorporated in different food formulas (fermented milk, fruit
juice) demonstrated significant decrease in blood pressure (SBP and DBP) in Japanese and Finnish
hypertensive volunteers [140,141]. However, the oral intake of these same peptides failed to reduce BP
in Dutch and Danish hypertensive subjects, suggesting possible differences in efficacy among different
human populations [142]. A meta-analysis of 18 clinical trials has shown that oral administration of
these peptides (VPP and IPP) does reduce BP in hypertensive subjects but the beneficial effect appears
to be pronounced in Asian subjects [143]. The controversial results obtained by different studies about
the effect of lactotripeptides in Caucasian population was addressed by Boelsma et al. in a double
blinded placebo control trial with 70 Caucasian pre-hypertensive or stage-1 hypertensive subjects.
The result from this study reveals that oral administration of IPP exerts clinically relevant BP reduction
Int. J. Mol. Sci. 2015, 16 271

in Caucasian subjects with stage-1 hypertension [96]. Furthermore, another study has demonstrated
that administration of milk tri-peptides along with plant sterols can exhibit a clinically significant reduction
in SBP as well as serum total and low density lipoprotein (LDL)-cholesterol without adverse effects
in hypertensive, hypercholesterolemic subjects in a randomized, placebo-controlled double-blind
intervention [144]. Results from all these studies suggested that the reduction of blood pressure has
been observed after at least 1–2 weeks of treatment with maximum effect of 13 mm Hg for SBP and 8 mm
Hg for DBP with a dose range of approximately 3–55 mg/day. A recent meta-analysis showed that small
doses (2.0–10.2 mg/day) of milk casein derived tri-peptides (VPP and IPP) exhibited an overall reduction
of SBP by 4.0 mm Hg and DBP by 1.9 mm Hg in mildly hypertensive subjects [145]. Another clinical
trial conducted by Hirota et al. with 24 mildly hypertensive subjects showed that these two lactopeptides
(VPP and IPP) also improves endothelial dysfunction and significantly increase hyperemia measured
on the left upper forearm of the subjects [146]. In addition to VPP and IPP, another study showed that
consumption of yogurt enriched with casein derived antihypertensive peptides (RYLGY and AYFYPEL)
reduced significantly SBP by 12 mmHg after 6 weeks of intake in a normalized placebo control trial [29].
Human clinical trials with pea protein hydrolysate also showed significant decrease in SBP by 5–7 mm
Hg after 2 weeks treatment, but the smaller number (n = 7) used in this study is obviously not enough
to judge the efficacy of the hydrolysate [147]. Similarly, sardine muscle derived di-peptide (VY) was
used for human clinical trials. A randomized double-blind placebo control trials with 29 hypertensive
subjects have demonstrated decrease in SBP and DBP by 9.3 and 5.2 mmHg, respectively, after 4 weeks
of treatment [148]. However the clinical impact is controversial due to the small number of heterogeneous
subjects in the trials [39]. Another trial involving 63 hypertensive subjects have shown that consumption
of a vegetable drink containing VY could significantly reduce the blood pressure in high and mild
hypertensive subjects, without any adverse side effects [149]. The third clinical study showed that
a single oral administration of VY could significantly increase the plasma VY level, indicating the
absorption of peptide in the blood stream. However no marked decrease in blood pressure was observed
with the increased plasma VY level, indicating that VY did not exhibit an acute anti-hypertensive
effect after oral ingestion and a longer-term treatment was likely necessary for the clinical benefits [150].
Various animal studies showed BP reduction by 20–40 mmHg using food protein derived bioactive
peptides [29,99,115,118]; however, studies on human subjects were limited and the extent of blood
pressure reduction was much less, mostly by 2–12 mmHg [142,143,151]. It should be noted that most
animal studies were performed in a specific model of hypertension (such as SHR) where all animals
had the same particular pathophysiology but the human subjects were likely to have different etiology
underlying their clinical hypertension. In addition, animal studies use specific strains of animals while
a group of human subjects in a clinical study are likely to have diverse racial and genetic backgrounds,
which might complicate the efficacy of bioactive peptides. Indeed racial and genetic factors are
known to modulate the development of hypertension [152,153]. Moreover, pharmacokinetics and
pharmacodynamics of these food-derived bioactive peptides could be different in rodents and humans
further complicating the efficacy of bioactive peptides. Therefore, more clinical studies engaging
the volunteers from various ethnicities are required to establish the efficacy of food derived bioactive
peptides as clinically relevant antihypertensive agents (Table 2).
Int. J. Mol. Sci. 2015, 16 272

Table 2. Human clinical trials of food protein derived antihypertensive peptides.

Active Duration
Administered Product Study Description Dose/Day SBP Decrease (mmHg) References
Peptide (Weeks)
Double-blinded placebo-controlled
150 mL (3.0 mg VPP and
Fermented milk randomized trial, 46 men with 21 −5.2 mm Hg [144]
2.25 mg IPP/100 g)
high-normal blood pressure.
Evolus® (fermented milk Placebo-controlled randomized trial,
160 g 4 −6.7 mm Hg [143]
flavored with fruit juice) 42 subjects with mild hypertension.
Randomized double-blind No significant difference in blood
200 mL (5.8 mg VPP and
Low-fat yoghurt drinks placebo-controlled trial, 135 hypertensive 8 pressure between the treatment [145]
5.4 mg IPP)
subjects (male/female: 88/47). and placebo controlled group
Placebo control, double blinded,
VPP and IPP 2-tablets/day (each tablet −4.0 mm Hg in SBP (significant
Milk protein hydrolysate crossover including 70 4 [97]
contains 7.5 mg IPP) reduction) No change in DBP
Caucasian subjects.
Randomized double blinded,
Fruit Juice fortified with 25 mL/day (3.0 mg of
52 (men:women = 29:21) mildly 6 −5.0 mm Hg in SBP [154]
Lacto tri-peptides VPP and IPP)
hypertensive patients.
Randomised, placebo-controlled
A lacto spread 20 g/day (containing −4.1 mm Hg in SBP, No change
double-blind intervention,
contained VPP, IPP and 4.2 mg of VPP and IPP; 10 in DBP and significantly reduce [147]
104 hypertensive,
plant sterols 2 g of plant sterols) plasma LDL cholesterol
hypercholesterolemic subjects.
RYLGY and 20 mL/day (5.5 mg of
Casein hydrolysate Normalized placebo control trial. 6 −12 mm Hg in SBP [29]
AYFYPEL) RYLGY and AYFYPEL)
A beverage
Randomized placebo-controlled trial,
enriched with sardine 2 × 100 mL (6 mg VY) 4 −9.3 mm Hg [151]
29 subjects with mild hypertension.
muscle hydrolysate
VY
Randomized placebo-controlled trial,
A vegetable drink 63 subjects (male/female: 51/12) with 195 g (0.5 g VY) 13 −7.6 mm Hg [152]
mild hypertension.
Int. J. Mol. Sci. 2015, 16 273

5. General Conclusions

There is a tremendous global interest in promoting the use of food proteins/peptides as novel
alternatives for present pharmaceutical therapeutics in the treatment and prevention of high blood
pressure. In addition to exerting in vivo efficacy as the antihypertensive agent, food protein derived
bioactive peptides can interact with the various blood pressure regulatory pathways, indicating
their potential roles in controlling other pathologies related to the cardiovascular system, which
may represent the advantage of using bioactive peptides as functional food/nutraceutical ingredients.
However for many peptides, the actual mechanisms of action are not fully elucidated. Therefore,
further research is required to identify the molecular targets of peptide action, which is important to
establish the health promoting effects of these bioactive peptides. In this context, the use of advanced
biochemical technologies such as proteomics, RNA sequencing, computational study with molecular
docking and gene functional analysis are important to unlock the molecular mechanisms. Various clinical
studies involving volunteers from different ethnic groups are also required to evaluate the ultimate
efficacy and the pharmacokinetics of the active peptides. Furthermore, the safety of these bioactive
peptides should also be evaluated before commercialization. Concomitant long-term research is also
required to study the adverse or toxic effects associated with these active peptides. The translation of
food derived bioactive peptide for human health improvements is an exciting scientific challenge,
but simultaneously offers the opportunity for successful commercial applications. Lastly, various food
materials with high protein content as well as the byproducts of the food processing industries could be
used as a raw material for the industrial production of pharmaceutical grade bioactive peptides that
may result in reduction in the production cost and also provides a sustainable and effective way of handling
waste materials.

Acknowledgments

The laboratory of Jianping Wu is supported by Grants from Natural Sciences and Engineering
Research Council (NSERC) of Canada and Alberta Livestock and Meat Agency (ALMA).

Author Contributions

Conceived and designed the idea for the manuscript: Kaustav Majumder; Wrote the manuscript:
Kaustav Majumder and Jianping Wu.

Abbreviation

ACE 2: Angiotensin converting enzyme 2; ACE: Angiotensin I converting enzyme; Ang I:

Angiotensin I; Ang II: Angiotensin II; APoE: Apolipoprotein E; BP: Blood pressure; cGMP: cyclic
guanosine-monophosphate; CVDs: Cardiovascular diseases; DBP: Diastolic blood pressure;
ECE: endothelin converting enzyme; ECs: Endothelial cells; EGFs: Epidermal growth factors;
eNOS: Endothelial nitric oxide synthase; ET-1: Endothelin; GPCR: g-protein coupled receptor;
ICAM-1: Intercellular adhesion molecule 1; IL: interleukin; iNOS: inducible nitric oxide synthase;
KKS: Kallikrein kinin–system; MAP: Mean arterial pressure; MCP-1: Monocyte chemotactic
protein-1; MMP: metalloproteinases; NADPH: nicotinamide adenine dinucleotide phosphate;
Int. J. Mol. Sci. 2015, 16 274

NEP: neutral endopeptidase; NO: Nitric oxide; PGI2: Prostacyclin; RAS: Renin angiotensin system;
ROS: Reactive oxygen species; SBP: Systolic blood pressure; sGC: soluble guanylyl cyclase; TNF-α:
Tumor necrosis factor-α; VCAM-1: Vascular adhesion molecule-1; VSM: Vascular smooth muscle.

Conflicts of Interest

The authors declare no conflict of interest.

References

1. WHO. A Global Brief on Hypertension, Silent Killer, Global Public Health Crisis; WHO:
Geneva, Switzerland, 2013.
2. Public Health Agency of Canada. Tracking Heart Disease and Stroke in Canada—Stroke
Highlights 2011; Public Health Agency of Canada: Ottawa, ON, Canada, 2011.
3. Moser, M.; Franklin, S.S. Hypertension Management: Results of a New National Survey for the
Hypertension Education Foundation: Harris Interactive. J. Clin. Hypertens. (Greenwich) 2007, 9,
316–323.
4. Williams, B.; Poulter, N.R.; Brown, M.J.; Davis, M.; McInnes, G.T.; Potter, J.F.; Sever, P.S.;
Thom, S.M. Guidelines for Management of Hypertension: Report of the Fourth Working Party of
the British Hypertension Society, 2004—BHSIV. J. Hum. Hypertens. 2004, 18, 139–185.
5. Klag, M.J.; Whelton, P.K.; Randall, B.L.; Neaton, J.D.; Brancati, F.L.; Ford, C.E.; Shulman, N.B.;
Stamler, J. Blood Pressure and End-Stage Renal Disease in Men. N. Engl. J. Med. 1996, 334,
13–18.
6. De las Heras, N.; Ruiz-Ortega, M.; Ruperez, M.; Sanz-Rosa, D.; Miana, M.; Aragoncillo, P.;
Mezzano, S.; Lahera, V.; Egido, J.; Cachofeiro, V. Role of Connective Tissue Growth Factor
in Vascular and Renal Damage Associated with Hypertension in Rats. Interactions with
Angiotensin II. J. Renin Angiotensin Aldosterone Syst. 2006, 7, 192–200.
7. Perazella, M.A.; Setaro, J.F. Renin-Angiotensin-Aldosterone System: Fundamental Aspects and
Clinical Implications in Renal and Cardiovascular Disorders. J. Nucl. Cardiol. 2003, 10, 184–196.
8. Alexander, R.W. Hypertension and the Pathogenesis of Atherosclerosis: Oxidative Stress and the
Mediation of Arterial Inflammatory Response: A New Perspective. Hypertension 1995, 25,
155–161.
9. Altman, R. Risk Factors in Coronary Atherosclerosis Athero-Inflammation: The Meeting Point.
Thromb. J. 2003, 1, 4.
10. US Department of Health & Human Services. HHS Secretary Sebelius Statement on National
High Blood Pressure Education Month; US Department of Health & Human Services:
Washington, DC, USA, 2012.
11. Atkinson, A.B.; Robertson, J.I.S. Captopril in the Treatment of Clinical Hypertension and
Cardiac-Failure. Lancet 1979, 2, 836–839.
12. Gavras, H.; Gavras, I. Angiotensin Converting Enzyme Inhibitors. Properties and Side Effects.
Hypertension 1988, 11, II37.
Int. J. Mol. Sci. 2015, 16 275

13. Chobanian, A.V.; Bakris, G.L.; Black, H.R.; Cushman, W.C.; Green, L.A.; Izzo, J.L.; Jones, D.W.;
Materson, B.J.; Oparil, S.; Wright, J.T.; et al. Seventh Report of the Joint National Committee on
Prevention, Detection, Evaluation, and Treatment of High Blood Pressure. Hypertension 2003, 42,
1206–1252.
14. Vaughan, C.J.; Delanty, N. Hypertensive Emergencies. Lancet 2000, 356, 411–417.
15. Stephanie, J.; Suhad, S.A.; Peter, J.H.J. Evolution of the Human Diet: Linking our Ancestral Diet
to Modern Functional Foods as a Means of Chronic Disease Prevention. J. Med. Food 2009, 12,
925–934.
16. Miguel, M.G.; Maria, B.R.; Lluis, S.M.; Denis, L.; Ramón, E.; Antonia, T. Mediterranean Food
Pattern and the Primary Prevention of Chronic Disease: Recent Developments. Nutr. Rev. 2009,
67, S111–S116.
17. Mozaffarian, D.; Hao, T.; Rimm, E.B.; Willett, W.C.; Hu, F.B. Changes in Diet and Lifestyle and
Long-Term Weight Gain in Women and Men. N. Engl. J. Med. 2011, 364, 2392–2404.
18. Craddick, S.R.; Elmer, P.J.; Obarzanek, E.; Vollmer, W.M.; Svetkey, L.P.; Swain, M.C. The DASH
Diet and Blood Pressure. Curr. Atheroscler. Rep. 2003, 5, 484–491.
19. Rouse, I.; Armstrong, B.; Beilin, L.J.; Vandongen, R. Blood-Pressure-Lowering Effect of a
Vegetarian Diet: Controlled Trial in Normotensive Subjects. Lancet 1983, 321, 5–10.
20. Doaei, S.; Gholamalizadeh, M. The Association of Genetic Variations with Sensitivity of Blood
Pressure to Dietary Salt: A Narrative Literature Review. ARYA Atheroscler. 2014, 10, 169–174.
21. Stolarz-Skrzypek, K.; Bednarski, A.; Czarnecka, D.; Kawecka-Jaszcz, K.; Staessen, J.A.
Sodium and Potassium and the Pathogenesis of Hypertension. Curr. Hypertens. Rep. 2013, 15,
122–130.
22. Aaron, K.J.; Sanders, P.W. Role of Dietary Salt and Potassium Intake in Cardiovascular Health
and Disease: A Review of the Evidence. Mayo Clin. Proc. 2013, 88, 987–995.
23. Rebholz, C.M.; Friedman, E.E.; Powers, L.J.; Arroyave, W.D.; He, J.; Kelly, T.N. Dietary Protein
Intake and Blood Pressure: A Meta-Analysis of Randomized Controlled Trials. Am. J. Epidemiol.
2012, 176 (Suppl. S7), S27–S43.
24. Carey, V.J.; Bishop, L.; Charleston, J.; Conlin, P.; Erlinger, T.; Laranjo, N.; McCarron, P.;
Miller, E.; Rosner, B.; Swain, J.; et al. Rationale and Design of the Optimal Macro-Nutrient
Intake Heart Trial to Prevent Heart Disease (OMNI-Heart). Clin. Trials 2005, 2, 529–537.
25. Miller, E.R., 3rd; Erlinger, T.P.; Appel, L.J. The Effects of Macronutrients on Blood Pressure
and Lipids: An Overview of the DASH and OmniHeart Trials. Curr. Atheroscler. Rep. 2006, 8,
460–465.
26. Hartmann, R.; Meisel, H. Food-Derived Peptides with Biological Activity: From Research to
Food Applications. Curr. Opin. Biotechnol. 2007, 18, 163–169.
27. Miguel, M.; Aleixandre, A. Anti Hypertensive Peptides Derived from Egg Proteins. J. Nutr.
2006, 136, 1457–1460.
28. Torruco-Uco, J.G.; Dominguez-Magana, M.A.; Davila-Ortiz, G.; Martinez-Ayala, A.;
Chel-Guerrero, L.A.; Betancur-Ancona, D.A. Antihypertensive Peptides, an Alternative for
Treatment of Natural Origin: A Review. Cienc. Tecnol. Aliment. 2008, 6, 158–168.
29. Martinez-Maqueda, D.; Miralles, B.; Recio, I.; Hernandez-Ledesma, B. Antihypertensive Peptides
from Food Proteins: A Review. Food Funct. 2012, 3, 350–361.
Int. J. Mol. Sci. 2015, 16 276

30. Peah, M. Renin-Angiotensin System—Biochemistry and Mechanisms of Action. Physiol. Rev.

1977, 57, 313–370.
31. Zaman, M.; Oparil, S.; Calhoun, D. Drugs Targeting the Renin-Angiotensin-Aldosterone System.
Nat. Rev. Drug Discov. 2002, 1, 621–636.
32. Akpaffiong, M.J.; Taylor, A.A. Antihypertensive and Vasodilator Actions of Antioxidants in
Spontaneously Hypertensive Rats. Am. J. Hypertens. 1998, 11, 1450–1460.
33. De Ciuceis, C.; Porteri, E.; Rizzoni, D.; Rezzani, R.; Rodella, L.F.; Boari, G.E.; Tengattini, S.;
Bonomini, F.; Rizzardi, N.; Platto, C.; et al. Antioxidant Treatment with Melatonin and
Pycnogenol may Improve the Structure and Function of Mesenteric Small Arteries in Spontaneously
Hypertensive Rats. Hypertension 2009, 54, E119–E119.
34. Davalos, A.; Miguel, M.; Bartolome, B.; Lopez-Fandino, R. Antioxidant Activity of Peptides
Derived from Egg White Proteins by Enzymatic Hydrolysis. J. Food Prot. 2004, 67, 1939–1944.
35. Cheung, I.W.Y.; Nakayama, S.; Hsu, M.N.K.; Samaranayaka, A.G.P.; Li-Chan, E.C.Y.
Angiotensin-I Converting Enzyme Inhibitory Activity of Hydrolysates from Oat (Avena Sativa)
Proteins by in Silico and in Vitro Analyses. J. Agric. Food Chem. 2009, 57, 9234–9242.
36. Miguel, M.; Recio, I.; Gomez-Ruiz, J.A.; Ramos, M.; Lopez-Fandino, R. Angiotensin I-Converting
Enzyme Inhibitory Activity of Peptides Derived from Egg White Proteins by Enzymatic Hydrolysis.
J. Food Prot. 2004, 67, 1914–1920.
37. Maeno, M.; Yamamoto, N.; Takano, T. Identification of an Antihypertensive Peptide from Casein
Hydrolysate Produced by a Proteinase from Lactobacillus Helveticus CP790. J. Dairy Sci.
1996, 79, 1316–1321.
38. Hideaki, K.; Kunio, D.; Shigeru, S.; Hideyo, U.; Ryuji, S.; Umeji, M.; Shizume, T.
Antihypertensive Effect of Tryptic Hydrolysate of Milk Casein in Spontaneously Hypertensive
Rats. Comp. Biochem. Physiol. Part C Comp. Pharmacol. 1990, 96, 367–371.
39. Marques, C.; Amorim, M.M.; Pereira, J.O.; Pintado, M.E.; Moura, D.; Calhau, C.; Pinheiro, H.
Bioactive Peptides: Are there More Antihypertensive Mechanisms Beyond ACE Inhibition?
Curr. Pharm. Des. 2012, 18, 4706–4713.
40. Udenigwe, C.C.; Mohan, A. Mechanisms of Food Protein-Derived Antihypertensive Peptides
Other than ACE Inhibition. J. Funct. Foods 2014, 8, 45–52.
41. Siro, I.; Kapolna, E.; Kapolna, B.; Lugasi, A. Functional Food. Product Development, Marketing
and Consumer Acceptance—A Review. Appetite 2008, 51, 456–467.
42. Matsui, T.; Tanaka, M. Antihypertensive Peptides and Their Underlying Mechanisms;
Anonymous, Ed.; Wiley-Blackwell: Oxford, UK, 2010; pp. 43–54.
43. Viera, A.J.; Neutze, D.M. Diagnosis of Secondary Hypertension: An Age-Based Approach.
Am. Fam. Phys. 2010, 82, 1471–1478.
44. Carretero, O.A.; Oparil, S. Essential Hypertension: Part I: Definition and Etiology. Circulation
2000, 101, 329–335.
45. Schulz, E.; Gori, T.; Munzel, T. Oxidative Stress and Endothelial Dysfunction in Hypertension.
Hypertens. Res. 2011, 34, 665–673.
46. Paravicini, T.M.; Touyz, R.M. Redox Signaling in Hypertension. Cardiovasc. Res. 2006, 71,
247–258.
Int. J. Mol. Sci. 2015, 16 277

47. Cuzzocrea, S.; Mazzon, E.; Dugo, L.; di Paola, R.; Caputi, A.P.; Salvemini, D. Superoxide: A
Key Player in Hypertension. FASEB J. 2004, 18, 94–101.
48. Intengan, H.D.; Schiffrin, E.L. Vascular Remodeling in Hypertension: Roles of Apoptosis,
Inflammation, and Fibrosis. Hypertension 2001, 38, 581–587.
49. Hall, J.E.; Granger, J.P.; do Carmo, J.M.; da Silva, A.A.; Dubinion, J.; George, E.; Hamza, S.;
Speed, J.; Hall, M.E. Hypertension: Physiology and Pathophysiology. Comp. Physiol. 2012, 2,
2393–2442.
50. Foëx, P.; Sear, J. Hypertension: Pathophysiology and Treatment. Continuing Education in
Anaesthesia. Crit. Care Pain 2004, 4, 71–75.
51. Beevers, G.; Lip, G.Y.H.; O’Brien, E. The Pathophysiology of Hypertension. BMJ 2001, 322,
912–916.
52. Oparil, S.; Haber, E. The Renin-Angiotensin System. N. Engl. J. Med. 1974, 291, 389–401.
53. Zhuo, J.L.; Ferrao, F.M.; Zheng, Y.; Li, X.C. New Frontiers in the Intrarenal Renin-Angiotensin
System: A Critical Review of Classical and New Paradigms. Front. Endocrinol. (Lausanne)
2013, 4, 166.
54. Bader, M.; Ganten, D. Update on Tissue Renin-Angiotensin Systems. J. Mol. Med. 2008, 86,
615–621.
55. Michel, J.B. Renin-Angiotensin System and Vascular Remodelling. Med. Sci. (Paris) 2004, 20,
409–413.
56. Sriramula, S.; Cardinale, J.P.; Lazartigues, E.; Francis, J. ACE2 Overexpression in the
Paraventricular Nucleus Attenuates Angiotensin II-Induced Hypertension. Cardiovasc. Res.
2011, 92, 401–408.
57. Liu, K.L. Regulation of Renal Medullary Circulation by the Renin-Angiotensin System in
Genetically Hypertensive Rats. Clin. Exp. Pharmacol. Physiol. 2009, 36, 455–461.
58. Millatt, L.J.; Abdel-Rahman, E.M.; Siragy, H.M. Angiotensin II and Nitric Oxide: A Question of
Balance. Regul. Pept. 1999, 81, 1–10.
59. Stankevicius, E.; Kevelaitis, E.; Vainorius, E.; Simonsen, U. Role of Nitric Oxide and Other
Endothelium-Derived Factors. Medicina (Kaunas) 2003, 39, 333–341.
60. Danyel, L.A.; Schmerler, P.; Paulis, L.; Unger, T.; Steckelings, U.M. Impact of AT2-Receptor
Stimulation on Vascular Biology, Kidney Function, and Blood Pressure. Integr. Blood Press Control.
2013, 6, 153–161.
61. Nicholls, M. Inhibition of the Renin-Angiotensin System in the Treatment of Heart-Failure—Why,
when, and Where. J. Cardiovasc. Pharmacol. 1985, 7, S98–S102.
62. Reaves, P.Y.; Beck, C.R.; Wang, H.W.; Raizada, M.K.; Katovich, M.J. Endothelial-Independent
Prevention of High Blood Pressure in L-NAME-Treated Rats by Angiotensin II Type I Receptor
Antisense Gene Therapy. Exp. Physiol. 2003, 88, 467–473.
63. Schmaier, A.H. The Kallikrein-Kinin and the Renin-Angiotensin Systems have a Multilayered
Interaction. Am. J. Physiol. Regul. Integr. Comp. Physiol. 2003, 285, R1–R13.
64. Behrendt, D.; Ganz, P. Endothelial Function from Vascular Biology to Clinical Applications.
Am. J. Cardiol. 2002, 90, 40L–48L.
65. Pober, J.S.; Min, W.; Bradley, J.R. Mechanisms of Endothelial Dysfunction, Injury, and Death.
Annu. Rev. Pathol. Mech. Dis. 2009, 4, 71–95.
Int. J. Mol. Sci. 2015, 16 278

66. Aroor, A.R.; Demarco, V.G.; Jia, G.; Sun, Z.; Nistala, R.; Meininger, G.A.; Sowers, J.R.
The Role of Tissue Renin-Angiotensin-Aldosterone System in the Development of Endothelial
Dysfunction and Arterial Stiffness. Front. Endocrinol. (Lausanne) 2013, 4, 161.
67. Zhang, H.; Park, Y.; Wu, J.; Chen, X.; Lee, S.; Yang, J.; Dellsperger, K.C.; Zhang, C. Role of
TNF-Alpha in Vascular Dysfunction. Clin. Sci. (Lond.) 2009, 116, 219–230.
68. Capettini, L.S.; Cortes, S.F.; Lemos, V.S. Relative Contribution of eNOS and nNOS to
Endothelium-Dependent Vasodilation in the Mouse Aorta. Eur. J. Pharmacol. 2010, 643, 260–266.
69. Forstermann, U.; Sessa, W.C. Nitric Oxide Synthases: Regulation and Function. Eur. Heart J.
2012, 33, 829–837, 837a–837d.
70. Cohen, R.A.; Vanhoutte, P.M. Endothelium-Dependent Hyperpolarization. Beyond Nitric Oxide
and Cyclic GMP. Circulation 1995, 92, 3337–3349.
71. Sudano, I.; Spieker, L.E.; Hermann, F.; Flammer, A.; Corti, R.; Noll, G.; Luscher, T.F.
Protection of Endothelial Function: Targets for Nutritional and Pharmacological Interventions.
J. Cardiovasc. Pharmacol. 2006, 47 (Suppl. S2), S136–S150; discussion S172–S176.
72. Pennathur, S.; Heinecke, J.W. Oxidative Stress and Endothelial Dysfunction in Vascular Disease.
Curr. Diabetes Rep. 2007, 7, 257–264.
73. Nathan, C.; Xie, Q.W. Regulation of Biosynthesis of Nitric Oxide. J. Biol. Chem. 1994, 269,
13725–13728.
74. Barton, M.; Yanagisawa, M. Endothelin: 20 Years from Discovery to Therapy. Can. J.
Physiol. Pharmacol. 2008, 86, 485–498.
75. Bourque, S.L.; Davidge, S.T.; Adams, M.A. The Interaction between Endothelin-1 and Nitric Oxide
in the Vasculature: New Perspectives. Am. J. Physiol. Regul. Integr. Comp. Physiol. 2011, 300,
R1288–R1295.
76. Alonso, D.; Radomski, M. The Nitric Oxide-Endothelin-1 Connection. Heart Fail. Rev. 2003, 8,
107–115.
77. Mark, A.L. The Sympathetic Nervous System in Hypertension: A Potential Long-Term
Regulator of Arterial Pressure. J. Hypertens. Suppl. 1996, 14, S159–S165.
78. Tsuru, H.; Tanimitsu, N.; Hirai, T. Role of Perivascular Sympathetic Nerves and Regional
Differences in the Features of Sympathetic Innervation of the Vascular System. Jpn. J. Pharmacol.
2002, 88, 9–13.
79. Currie, G.; Freel, E.M.; Perry, C.G.; Dominiczak, A.F. Disorders of Blood Pressure Regulation-Role
of Catecholamine Biosynthesis, Release, and Metabolism. Curr. Hypertens. Rep. 2012, 14, 38–45.
80. Prabhakar, N.R.; Peng, Y.J.; Kumar, G.K.; Nanduri, J.; di Giulio, C.; Lahiri, S. Long-Term
Regulation of Carotid Body Function: Acclimatization and Adaptation—Invited Article.
Adv. Exp. Med. Biol. 2009, 648, 307–317.
81. Renna, N.F.; de Las Heras, N.; Miatello, R.M. Pathophysiology of Vascular Remodeling in
Hypertension. Int. J. Hypertens. 2013, 2013, 808353.
82. Giannotti, G.; Landmesser, U. Endothelial Dysfunction as an Early Sign of Atherosclerosis.
Herz 2007, 32, 568–572.
83. Cheng, Z.J.; Vapaatalo, H.; Mervaala, E. Angiotensin II and Vascular Inflammation. Med. Sci. Monit.
2005, 11, RA194–RA205.
Int. J. Mol. Sci. 2015, 16 279

84. Visse, R.; Nagase, H. Matrix Metalloproteinases and Tissue Inhibitors of Metalloproteinases:
Structure, Function, and Biochemistry. Circ. Res. 2003, 92, 827–839.
85. Schmid-Schönbein, G.W. Matrix Metalloproteinases Activities in Hypertension: Emerging
Opportunities. Hypertension 2011, 57, 24–25.
86. Yasmin,; Wallace, S.; McEniery, C.M.; Dakham, Z.; Pusalkar, P.; Maki-Petaja, K.; Ashby, M.J.;
Cockcroft, J.R.; Wilkinson, I.B. Matrix Metalloproteinase-9 (MMP-9), MMP-2, and Serum Elastase
Activity are Associated with Systolic Hypertension and Arterial Stiffness. Arterioscler. Thromb.
Vasc. Biol. 2005, 25, 372–378.
87. Cheung, P.Y.; Sawicki, G.; Wozniak, M.; Wang, W.; Radomski, M.W.; Schulz, R.
Matrix Metalloproteinase-2 Contributes to Ischemia-Reperfusion Injury in the Heart. Circulation
2000, 101, 1833–1839.
88. Hao, L.; Du, M.; Lopez-Campistrous, A.; Fernandez-Patron, C. Agonist-Induced Activation of
Matrix Metalloproteinase-7 Promotes Vasoconstriction through the Epidermal Growth
Factor-Receptor Pathway. Circ. Res. 2004, 94, 68–76.
89. Neutel, J.M.; Smith, D.H. Hypertension Management: Rationale for Triple Therapy Based on
Mechanisms of Action. Cardiovasc. Ther. 2013, 31, 251–258.
90. Ferreira, S.H.; Bartelt, D.C.; Greene, L.J. Isolation of Bradykinin-Potentiating Peptides from
Bothrops Jararaca Venom. Biochemistry 1970, 9, 2583–2593.
91. Ondetti, M.A.; Williams, N.J.; Sabo, E.F.; Pluscec, J.; Weaver, E.R.; Kocy, O.
Angiotensin-Converting Enzyme Inhibitors from the Venom of Bothrops Jararaca. Isolation,
Elucidation of Structure, and Synthesis. Biochemistry 1971, 10, 4033–4039.
92. Gouda, K.G.M.; Gowda, L.R.; Rao, A.G.A.; Prakash, V. Angiotensin I-Converting Enzyme
Inhibitory Peptide Derived from Glycinin, the 11S Globulin of Soybean (Glycine Max). J. Agric.
Food Chem. 2006, 54, 4568–4573.
93. Shin, Z.I.; Yu, R.; Park, S.A.; Chung, D.K.; Ahn, C.W.; Nam, H.S.; Kim, K.S.; Lee, H.J.
His-His-Leu, an Angiotensin I Converting Enzyme Inhibitory Peptide Derived from Korean Soybean
Paste, Exerts Antihypertensive Activity in Vivo. J. Agric. Food Chem. 2001, 49, 3004–3009.
94. Miguel, M.; Manso, M.; Aleixandre, A.; Alonso, M.J.; Salaices, M.; Lopez-Fandino, R.
Vascular Effects, Angiotensin I-Converting Enzyme (ACE)-Inhibitory Activity, and Anti
Hypertensive Properties of Peptides Derived from Egg White. J. Agric. Food Chem. 2007, 55,
10615–10621.
95. Xu, J.Y.; Qin, L.Q.; Wang, P.Y.; Li, W.; Chang, C. Effect of Milk Tripeptides on Blood Pressure:
A Meta-Analysis of Randomized Controlled Trials. Nutrition 2008, 24, 933–940.
96. Boelsma, E.; Kloek, J. IPP-Rich Milk Protein Hydrolysate Lowers Blood Pressure in Subjects with
Stage 1 Hypertension, a Randomized Controlled Trial. Nutr. J. 2010, 9, 52.
97. Majumder, K.; Chakrabarti, S.; Morton, J.S.; Panahi, S.; Kaufman, S.; Davidge, S.T.; Wu, J.
Egg-Derived Tri-Peptide IRW Exerts Antihypertensive Effects in Spontaneously Hypertensive
Rats. PLoS One 2013, 8, e82829.
98. Balti, R.; Bougatef, A.; Guillochon, D.; Dhulster, P.; Nasri, M.; Nedjar-Arroume, N. Changes in
Arterial Blood Pressure After Single Oral Administration of Cuttlefish (Sepia Officinalis) Muscle
Derived Peptides in Spontaneously Hypertensive Rats. J. Funct. Foods 2012, 4, 611–617.
Int. J. Mol. Sci. 2015, 16 280

99. Miguel, M.; Gomez-Ruiz, J.A.; Recio, I.; Aleixandre, A. Changes in Arterial Blood Pressure
After Single Oral Administration of Milk-Casein-Derived Peptides in Spontaneously Hypertensive
Rats. Mol. Nutr. Food Res. 2010, 54, 1422–1427.
100. Masuda, O.; Nakamura, Y.; Takano, T. Antihypertensive Peptides are Present in Aorta after Oral
Administration of Sour Milk Containing these Peptides to Spontaneously Hypertensive Rats.
J. Nutr. 1996, 126, 3063–3068.
101. Yamada, A.; Sakurai, T.; Ochi, D.; Mitsuyama, E.; Yamauchi, K.; Abe, F. Novel Angiotensin
I-Converting Enzyme Inhibitory Peptide Derived from Bovine Casein. Food Chem. 2013, 141,
3781–3789.
102. Nakamura, Y.; Yamamoto, N.; Sakai, K.; Okubo, A.; Yamazaki, S.; Takano, T. Purification and
Characterization of Angiotensin I-Converting Enzyme Inhibitors from Sour Milk. J. Dairy Sci.
1995, 78, 777–783.
103. Nakamura, Y.; Yamamoto, N.; Sakai, K.; Takano, T. Antihypertensive Effect of Sour Milk and
Peptides Isolated from it that are Inhibitors to Angiotensin I-Converting Enzyme. J. Dairy Sci.
1995, 78, 1253–1257.
104. Sipola, M.; Finckenberg, P.; Korpela, R.; Vapaatalo, H.; Nurminen, M.L. Effect of Long-Term Intake
of Milk Products on Blood Pressure in Hypertensive Rats. J. Dairy Res. 2002, 69, 103–111.
105. Fujita, H.; Yoshikawa, M. LKPNM: A Prodrug-Type ACE-Inhibitory Peptide Derived from Fish
Protein. Immunopharmacology 1999, 44, 123–127.
106. Majumder, K.; Wu, J. A New Approach for Identification of Novel Antihypertensive Peptides
from Egg Proteins by QSAR and Bioinformatics. Food Res. Int. 2010, 43, 1371–1378.
107. Contreras, F.; de la Parte, M.A.; Cabrera, J.; Ospino, N.; Israili, Z.H.; Velasco, M. Role of
Angiotensin II AT1 Receptor Blockers in the Treatment of Arterial Hypertension. Am. J. Ther.
2003, 10, 401–408.
108. Fernandez-Musoles, R.; Castello-Ruiz, M.; Arce, C.; Manzanares, P.; Ivorra, M.D.; Salom, J.B.
Antihypertensive Mechanism of Lactoferrin-Derived Peptides: Angiotensin Receptor Blocking
Effect. J. Agric. Food Chem. 2014, 62, 173–181.
109. Yu, Z.; Yin, Y.; Zhao, W.; Chen, F.; Liu, J. Antihypertensive Effect of Angiotensin-Converting
Enzyme Inhibitory Peptide RVPSL on Spontaneously Hypertensive Rats by Regulating Gene
Expression of the Renin-Angiotensin System. J. Agric. Food Chem. 2014, 62, 912–917.
110. He, R.; Maloma S.A.; Girgih, A.T.; Ju, X.; Aluko, R.E. Glycinyl-histidinyl-serine (GHS), a novel
rapeseed protein-derived peptide has blood pressure-lowering effect in spontaneously hypertensive
rats. J. Agric. Food Chem. 2013, 61, 8396–8402.
111. Ehlers, P.I.; Nurmi, L.; Turpeinen, A.M.; Korpela, R.; Vapaatalo, H. Casein-Derived Tripeptide
Ile-Pro-Pro Improves Angiotensin-(1–7)- and Bradykinin-Induced Rat Mesenteric Artery Relaxation.
Life Sci. 2011, 88, 206–211.
112. Yamada, Y.; Matoba, N.; Usui, H.; Onishi, K.; Yoshikawa, M. Design of a Highly Potent
Anti-Hypertensive Peptide Based on Ovokinin(2–7). Biosci. Biotechnol. Biochem. 2002, 66,
1213–1217.
113. Miguel, M.; Alvarez, Y.; López-Fandiño, R.; Alonso, M.J.; Salaices, M. Vasodilator Effects of
Peptides Derived from Egg White Proteins. Regul. Pept. 2007, 140, 131–135.
Int. J. Mol. Sci. 2015, 16 281

114. Miguel, M.; Lopez-Fandino, R.; Ramos, M.; Aleixandre, A. Short-Term Effect of Egg-White
Hydrolysate Products on the Arterial Blood Pressure of Hypertensive Rats. Br. J. Nutr. 2005, 94,
731–737.
115. Yamaguchi, N.; Kawaguchi, K.; Yamamoto, N. Study of the Mechanism of Antihypertensive
Peptides VPP and IPP in Spontaneously Hypertensive Rats by DNA Microarray Analysis.
Eur. J. Pharmacol. 2009, 620, 71–77.
116. Sanchez, D.; Kassan, M.; Contreras Mdel, M.; Carron, R.; Recio, I.; Montero, M.J.; Sevilla, M.A.
Long-Term Intake of a Milk Casein Hydrolysate Attenuates the Development of Hypertension
and Involves Cardiovascular Benefits. Pharmacol. Res. 2011, 63, 398–404.
117. Hirota, T.; Nonaka, A.; Matsushita, A.; Uchida, N.; Ohki, K.; Asakura, M.; Kitakaze, M.
Milk Casein-Derived Tripeptides, VPP and IPP Induced NO Production in Cultured Endothelial
Cells and Endothelium-Dependent Relaxation of Isolated Aortic Rings. Heart Vessel. 2011, 26,
549–556.
118. Del Mar Contreras, M.; Carron, R.; Montero, M.J.; Ramos, M.; Recio, I. Novel Casein-Derived
Peptides with Antihypertensive Activity. Int. Dairy J. 2009, 19, 566–573.
119. Erdmann, K.; Grosser, N.; Schipporeit, K.; Schroder, H. The ACE Inhibitory Dipeptide Met-Tyr
Diminishes Free Radical Formation in Human Endothelial Cells Via Induction of Heme
Oxygenase-1 and Ferritin. J. Nutr. 2006, 136, 2148–2152.
120. Majumder, K.; Chakrabarti, S.; Davidge, S.T.; Wu, J. Structure and Activity Study of Egg
Protein Ovotransferrin Derived Peptides (IRW and IQW) on Endothelial Inflammatory Response
and Oxidative Stress. J. Agric. Food Chem. 2013, 61, 2120–2129.
121. Zhao, H.; Usui, H.; Ohinata, K.; Yoshikawa, M. Met-Arg-Trp Derived from Rubisco Lowers
Blood Pressure Via Prostaglandin D(2)-Dependent Vasorelaxation in Spontaneously Hypertensive
Rats. Peptides 2008, 29, 345–349.
122. Marczak, E.D.; Usui, H.; Fujita, H.; Yang, Y.; Yokoo, M.; Lipkowski, A.W.; Yoshikawa, M.
New Antihypertensive Peptides Isolated from Rapeseed. Peptides 2003, 24, 791–798.
123. Yamada, Y.; Ohinata, K.; Lipkowski, A.W.; Yoshikawa, M. Rapakinin, Arg-Ile-Tyr, Derived
from Rapeseed Napin, shows Anti-Opioid Activity via the Prostaglandin IP Receptor Followed
by the Cholecystokinin CCK(2) Receptor in Mice. Peptides 2011, 32, 281–285.
124. Lüscher, T.F.; Barton, M. Endothelins and Endothelin Receptor Antagonists: Therapeutic
Considerations for a Novel Class of Cardiovascular Drugs. Circulation 2000, 102, 2434–2440.
125. Maes, W.; van Camp, J.; Vermeirssen, V.; Hemeryck, M.; Ketelslegers, J.M.; Schrezenmeir, J.;
van Oostveldt, P.; Huyghebaert, A. Influence of the lactokinin Ala-Leu-Pro-Met-His-Ile-Arg
(ALPMHIR) on the release of endothelin-1 by endothelial cells. Regul. Pept. 2004, 118, 105–109.
126. Fernández-Musoles, R.; López-Díez, J.J.; Torregrosa, G.; Vallés, S.; Alborch, E.; Manzanares, P.;
Salom, J.B. Lactoferricin B-Derived Peptides with Inhibitory Effects on ECE-Dependent
Vasoconstriction. Peptides 2010, 31, 1926–1933.
127. Fernández-Musoles, R.; Salom, J.B.; Martínez-Maqueda, D.; López-Díez, J.J.; Recio, I.;
Manzanares, P. Antihypertensive Effects of Lactoferrin Hydrolyzates: Inhibition of Angiotensin-
and Endothelin-Converting Enzymes. Food Chem. 2013, 139, 994–1000.
128. Tanaka, M.; Matsui, T.; Ushida, Y.; Matsumoto, K. Vasodilating Effect of Di-Peptides in Thoracic
Aortas from Spontaneously Hypertensive Rats. Biosci. Biotechnol. Biochem. 2006, 70, 2292–2295.
Int. J. Mol. Sci. 2015, 16 282

129. Matsui, T.; Hayashi, A.; Tamaya, K.; Matsumoto, K.; Kawasaki, T.; Murakami, K.; Kimoto, K.
Depressor Effect Induced by Dipeptide, Val-Tyr, in Hypertensive Transgenic Mice is due, in Part, to
the Suppression of Human Circulating Renin-Angiotensin System. Clin. Exp. Pharmacol. Physiol.
2003, 30, 262–265.
130. Matsui, T.; Imamura, M.; Oka, H.; Osajima, K.; Kimoto, K.; Kawasaki, T.; Matsumoto, K.
Tissue Distribution of Antihypertensive Dipeptide, Val-Tyr, After its Single Oral Administration
to Spontaneously Hypertensive Rats. J. Pept. Sci. 2004, 10, 535–545.
131. Tanaka, M.; Tokuyasu, M.; Matsui, T.; Matsumoto, K. Endothelium-independent vasodilation
effect of Di- and Tri-peptides in thoracic aorta of sprague-dawley rats. Life Sci. 2008, 82, 869–875.
132. Wang, Z.; Watanabe, S.; Kobayashi, Y.; Tanaka, M.; Matsui, T. Trp-His, a Vasorelaxant Di-Peptide,
can Inhibit Extracellular Ca2+ Entry to Rat Vascular Smooth Muscle Cells through Blockade of
Dihydropyridine-Like L-Type Ca2+ Channels. Peptides 2010, 31, 2060–2066.
133. Feuerstein, G.; Siren, A.L. The opioid peptides. A role in hypertension? Hypertension 1987, 9,
561–565.
134. Nurminen, M.L.; Sipola, M.; Kaarto, H.; Pihlanto-Leppala, A.; Piilola, K.; Korpela, R.;
Tossavainen, O.; Korhonen, H.; Vapaatalo, H. Alpha-lactorphin lowers blood pressure measured by
radiotelemetry in normotensive and spontaneously hypertensive rats. Life Sci. 2000, 66, 1535–1543.
135. Sipola, M.; Finckenberg, P.; Vapaatalo, H.; Pihlanto-Leppälä, A.; Korhonen, H.; Korpela, R.;
Nurminen, M. Α-Lactorphin and Β-Lactorphin Improve Arterial Function in Spontaneously
Hypertensive Rats. Life Sci. 2002, 71, 1245–1253.
136. Aihara, K.; Ishii, H.; Yoshida, M. Casein-Derived Tripeptide, Val-Pro-Pro (VPP), Modulates
Monocyte Adhesion to Vascular Endothelium. J. Atheroscler. Thromb. 2009, 16, 594–603.
137. Nakamura, T.; Hirota, T.; Mizushima, K.; Ohki, K.; Naito, Y.; Yamamoto, N.; Yoshikawa, T.
Milk-Derived Peptides, Val-Pro-Pro and Ile-Pro-Pro, Attenuate Atherosclerosis Development in
Apolipoprotein E-Deficient Mice: A Preliminary Study. J. Med. Food 2013, 16, 396–403.
138. Huang, W.; Chakrabarti, S.; Majumder, K.; Jiang, Y.; Davidge, S.T.; Wu, J. Egg-Derived Peptide
IRW Inhibits TNF-α-Induced Inflammatory Response and Oxidative Stress in Endothelial Cells.
J. Agric. Food Chem. 2010, 58, 10840–10846.
139. Geleijnse, J.M.; Engberink, M.F. Lactopeptides and Human Blood Pressure. Curr. Opin. Lipidol.
2010, 21, 58–63.
140. Mizushima, S.; Ohshige, K.; Watanabe, J.; Kimura, M.; Kadowaki, T.; Nakamura, Y.;
Tochikubo, O.; Ueshima, H. Randomized Controlled Trial of Sour Milk on Blood Pressure in
Borderline Hypertensive Men. Am. J. Hypertens. 2004, 17, 701–706.
141. Seppo, L.; Jauhiainen, T.; Poussa, T.; Korpela, R. A Fermented Milk High in Bioactive Peptides has
a Blood Pressure-Lowering Effect in Hypertensive Subjects. Am. J. Clin. Nutr. 2003, 77, 326–330.
142. Engberink, M.F.; Schouten, E.G.; Kok, F.J.; van Mierlo, L.A.J.; Brouwer, I.A.; Geleijnse, J.M.
Lactotripeptides show no Effect on Human Blood Pressure: Results from a Double-Blind
Randomized Controlled Trial. Hypertension 2008, 51, 399–405.
143. Cicero, A.F.G.; Gerocarni, B.; Laghi, L.; Borghi, C. Blood Pressure Lowering Effect of
Lactotripeptides Assumed as Functional Foods: A Meta-Analysis of Current Available Clinical
Trials. J. Hum. Hypertens. 2011, 25, 425–436.
Int. J. Mol. Sci. 2015, 16 283

144. Turpeinen, A.M.; Ikonen, M.; Kivimaki, A.S.; Kautiainen, H.; Vapaatalo, H.; Korpela, R.
A Spread Containing Bioactive Milk Peptides Ile-Pro-Pro and Val-Pro-Pro, and Plant Sterols has
Antihypertensive and Cholesterol-Lowering Effects. Food Funct. 2012, 3, 621–627.
145. Turpeinen, A.M.; Jarvenpaa, S.; Kautiainen, H.; Korpela, R.; Vapaatalo, H. Antihypertensive Effects
of Bioactive Tripeptides-a Random Effects Meta-Analysis. Ann. Med. 2013, 45, 51–56.
146. Hirota, T.; Ohki, K.; Kawagishi, R.; Kajikoto, Y.; Mizuno, S.; Nakamura, Y.; Kitakaze, M.
Casein Hydrolysate Containing the Antihypertensive Tripeptides Val-Pro-Pro and Ile-Pro-Pro
improves Vascular Endothelial Function Independent of Blood Pressure-Lowering Effects:
Contribution of the Inhibitory Action of Angiotensin-Converting Enzyme. Hypertens. Res.
2007, 30, 489–496.
147. Li, H.; Prairie, N.; Udenigwe, C.C.; Adebiyi, A.P.; Tappia, P.S.; Aukema, H.M.; Jones, P.J.;
Aluko, R.E. Blood Pressure Lowering Effect of a Pea Protein Hydrolysate in Hypertensive Rats
and Humans. J. Agric. Food Chem. 2011, 59, 9854–9860.
148. Kawasaki, T.; Seki, E.; Osajima, K.; Yoshida, M.; Asada, K.; Matsui, T.; Osajima, Y.
Antihypertensive Effect of Valyl-Tyrosine, a Short Chain Peptide Derived from Sardine Muscle
Hydrolyzate, on Mild Hypertensive Subjects. J. Hum. Hypertens. 2000, 14, 519–523.
149. Kawasaki, T.; Jun, C.J.; Fukushima, Y.; Kegai, K.; Seki, E.; Osajima, K.; Itoh, K.; Matsui, T.;
Matsumoto, K. Antihypertensive Effect and Safety Evaluation of Vegetable Drink with Peptides
Derived from Sardine Protein Hydrolysates on Mild Hypertensive, High-Normal and Normal
Blood Pressure Subjects. Fukuoka Igaku Zasshi 2002, 93, 208–218.
150. Matsui, T.; Tamaya, K.; Seki, E.; Osajima, K.; Matsumo, K.; Kawasaki, T. Absorption of Val-Tyr
with in Vitro Angiotensin I-Converting Enzyme Inhibitory Activity into the Circulating Blood
System of Mild Hypertensive Subjects. Biol. Pharm. Bull. 2002, 25, 1228–1230.
151. Flack, J.M.; Ference, B.A.; Levy, P. Should African Americans with Hypertension be Treated
Differently than non-African Americans? Curr. Hypertens. Rep. 2014, 16, 1–6.
152. Ferdinand, K.C.; Nasser, S.A. A Review of the Efficacy and Tolerability of Combination
Amlodipine/Valsartan in non-White Paitents with Hypertension. Am. J. Cardiovasc. Drugs 2013,
13, 301–313.
153. Cicero, A.F.; Rosticci, M.; Gerocarni, B.; Bacchelli, S.; Veronesi, M.; Strocchi, E.; Borghi, C.
Lactotripeptides Effect on Office and 24-H Ambulatory Blood Pressure, Blood Pressure Stress
Response, Pulse Wave Velocity and Cardiac Output in Patients with High-Normal Blood Pressure
or First-Degree Hypertension: A Randomized Double-Blind Clinical Trial. Hypertens. Res.
2011, 34, 1035–1040.
154. Kuba, K.; Imai, Y.; Ohto-Nakanishi, T.; Penninger, J.M. Trilogy of ACE2: A Peptidase in the
Renin-Angiotensin System, a SARS Receptor, and a Partner for Amino Acid Transporters.
Pharmacol. Ther. 2010, 128, 119–128.

© 2014 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/4.0/).